Innovative targets of the lncRNA-miR-mRNA network in response to low-dose aspirin in breast cancer patients

This study aimed to investigate innovative targets in breast cancer patients by considering the interaction of the lncRNA-miR-mRNA network in response to low-dose aspirin. The candidate miRs were first taken from the GEO and TCGA databases. Then, the candidate network was constructed using the high-throughput sequencing data. The expression levels of candidate targets were finally measured using Real-Time PCR in luminal A breast cancer patients undergoing aspirin (80 mg daily for three months) and non-aspirin groups during chemotherapy after surgery. The expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins were measured using the ELISA technique. 5 lncRNAs, 12 miRs, and 10 genes were obtained in the bioinformatic phase. A significant expression increase of the candidate tumor suppressor lncRNAs, miRs, and genes and a substantial expression decrease of the candidate onco-lncRNAs, oncomiRs, and oncogenes were achieved after the aspirin consumption. Unlike the non-aspirin group, the expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins were significantly decreased following aspirin consumption. The Kaplan–Meier analysis indicated a longer overall survival rate in the patients after aspirin consumption. Our results showed that the lncRNA-miR-mRNA network might be a significant target for aspirin; their expression changes may be a new strategy with potential efficacy for cancer therapy or prevention.

Enrichment analysis of DEGs. Gene Ontology (GO) analysis was performed by FunRich to evaluate the biological functions of DEGs. The pathways were specifically enriched by DEGs, including receptor binding, cell cycle, proliferation, transcription factor activity, serine/threonine kinase activity, growth factor activity, DNA repair protein, EGF receptor, glypican, ErbB, and Rap1 (Table 6). All candidate miRs could regulate the signaling pathways, including Wnt, PI3K-AKT, EGF, NOTCH, JAK/STAT, and apoptosis. The most modified routes were the PI3K/AKT and WNT pathways. Thus, candidate miRs could target the significant genes involved in these pathways ( Table 6). As shown in Fig. 2, functional enrichment of DEG genes was also analyzed using g: Profiler software.
The expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins. The expression levels of TGFβ, IL-17, IFNγ, and IL-β proteins were measured using the ELISA technique. TGFβ, IL-17, IFNγ, and IL-β expressions were significantly increased in the patients (pre-treatment) compared to the control group (P < 0.05). Unlike the non-aspirin group, the proteins' expression levels were significantly decreased following aspirin consumption; however, their expressions were significantly lower in aspirin users than in non-aspirin users (Table 7). www.nature.com/scientificreports/ The expression of onco-lncRNAs, oncomiRs, and oncogenes. The expression levels of the onco-lncRNAs ( Fig. 6A-C), -miRs ( Fig. 7A-H), and -mRNAs ( Fig. 8A-G) were significantly increased in the patients (pre-treatment) compared to the control group (P < 0.05). Unlike the non-aspirin group, the expression levels of the onco-lncRNAs, -miRs, and -mRNAs were significantly decreased following aspirin consumption (P < 0.0001).

Figure 1.
A Venn diagram of the differently expressed lncRNAs, miRs, and mRNAs between GEO and TCGA datasets. Allocation of (A) the 157 differently expressed miRs (37 up-regulation and 120 down-regulation), (B) 2183 differently expressed mRNAs (996 up-regulation and 1187 down-regulation), and (C) 169 differently expressed lncRNAs (102 up-regulation and 67 down-regulation) was found between the datasets in the present study. www.nature.com/scientificreports/ The expression of the tumor suppressor lncRNAs, miRs, and mRNAs. The expression levels of the tumor suppressor -lncRNAs (Fig. 6D,E), -miRs ( Fig. 7I-L), and -mRNAs ( Fig. 8H-J) were significantly decreased in the patients (pre-treatment) compared to the control group (P < 0.05). The tumor suppressor -lncR-NAs, -miRs, and -mRNAs were significantly higher in aspirin users than non-aspirin users (P < 0.0001). Table 2. Interaction analysis between selected miRs and target genes in breast cancer patients.

Discussion
In the present study, we measured the effects of aspirin consumption on the expression profiles of lncRNAs, miRs, and mRNAs in patients with non-metastatic early luminal A breast cancer. Using computational approaches, we first constructed a network of the lncRNA-miR-mRNA based on our multi-level methodology. 5 lncRNAs, 12 miRs, and 10 mRNAs have been shown to have significant differential expressions in cancerous tissues compared to non-cancerous tissues, improved after aspirin consumption. To our knowledge, this is the first study to demonstrate aspirin's effects on competing endogenous RNA (ceRNA; lncRNA-miRNA-mRNA) perturbation. They can be promising tools for early detection, prognosis, and monitoring treatment effects. Most of the retrieved biomarkers from our computational analyses have been shown to play essential roles in breast cancer. For instance, miR-21 can increase cell growth and metastasis 19 . MiR-20 family members may also contribute to cell proliferation, migration, invasion, and angiogenic reactions 20,21 . Similar to our results, other molecules such as HOTAIR 22 , XIST 23,24 , GAS5 25,26 , Zfas1 27 , miR-125a, miR-155 28 , miR-224 12 , miR-106a 29 , miR-17 30 , miR-141 31 , miR-145 32 , miR-200a 33 , miR-196b, miR-193b 34 , miR-196a 35 , miR-205 36 , and miR-342 37 , have been reported to play important roles in breast cancer. At the molecular level, ceRNAs can inhibit protein production by affecting the stability of their target mRNAs 38 . Moreover, a recent meta-analysis showed the converse correlation of the lncRNA levels with the risk of poor outcomes in breast cancer patients 39 . To our knowledge, the constructed network between these molecules has not been previously reported. It has been established that the cross-talk between lncRNAs and miRs can be a principal component of cancer pathophysiology 40 . In this regard, Zhang et al. (2017) constructed a massive network of lncRNA-miR-mRNA in breast cancer. Accordingly, miR-510 was the most potent miRNA controller and regulator of numerous target genes. Besides, they showed a group of lncRNAs, including PVT1, CCAT1, and linc00861, that interacted with particular clinical biomarkers such as estrogen and progesterone receptors 41 . In this study, we focused on aspirin effects on the constructed network expression. Aspirin could reduce the WNT activity, arresting the cell cycle via the WNT/β-catenin axis. In this setting, Khan et al. (2019) demonstrated that aspirin could inhibit the cell migration and invasion via the down-regulation of WNT/β-catenin, consequently reducing fibromodulin expression 42 . Besides, Tang et al. (2016) used aspirin and ursolic acid to co-treatment against breast cancer. This combination could reduce the metastatic feature of breast cancer via regulating EGFR mediating signaling pathways 43 . It was also shown that aspirin could increase the PI3K pathway inhibitors 44,45 . According to , PI3K pathway inhibitors had limited clinical response despite the high incidence of PIK3CA mutations in breast cancer patients 44 . They showed that aspirin selectively inhibited the growth of mutant PIK3CA breast cells. Besides, it could sensitize mutant PIK3CA cells to PI3K inhibitors. Co-treatment of aspirin and PI3K inhibitors are led to AMPK activation, mTORC1 inhibition, and autophagy induction 44 . Furthermore, Cheng et al. (2018) showed that aspirin-alone treatment reduced the proliferation of estrogen-positive breast cancer cells. Moreover, aspirin could increase the sensitivity of the tamoxifen-resistant breast cancer cells to tamoxifen by inhibiting c-Myc and cyclin D1 proteins 46 . Consistent results have also been reported in other cancers. Xie et al. (2018) showed that aspirin could enhance the sensitivity of hepatocellular carcinoma cells to doxorubicin via modulation of miR-491/ABCG2 expression 47 . Altogether, the findings of this study and those reported in the literature can support the idea of aspirin co-administration with chemotherapy regimens in breast cancer patients. However, these findings should be interpreted cautiously, and large-scale clinical trials should be conducted to assess the co-administration effects of aspirin with different chemotherapy agents in breast cancer patients.
As the well-known aspirin effects are anti-inflammatory, we measured a group of cytokines closely related to our constructed ceRNA network. In this respect, aspirin users had decreased levels of TGFβ, IFNγ, IL-1b, and IL-17. Similar to our findings,  observed that low-dose aspirin administration would reduce the COX2 and TGFβ intensity in breast cancer patients previously irradiated 48 . In the early stages of tumorigenesis, TGFβ1 acts as a tumor suppressor by inhibiting cell proliferation, inducing apoptosis, and suppressing growth factors, cytokine, and chemokine production. TGFβ1 overexpression can impair immune surveillance and promote angiogenesis, tumor invasion, and metastasis. Besides, aspirin can inflate the anti-tumoral effects of IFN-α. From a closer look, aspirin could enhance the IFN-α-induced apoptosis via the JAK1/STAT1 pathway 49 . Moreover, earlier studies revealed that the intra-tumoral levels of IL-17 were increased and correlated with the expansion of breast cancer 50   Clinical applications. Aspirin is one of the most widely used NSAIDs globally; remarkably, it is still one of the most attractive medicines globally, with an extent of use much beyond its primary usage in controlling fever, pain, and inflammation 51 . For example, the co-prescription of the drugs, such as clopidogrel and oral anticoagulation, with aspirin in populations with coronary artery diseases could reduce the risk of death and myocardial    www.nature.com/scientificreports/ infarction 52 . Moreover, a low dose of aspirin was recommended for pregnant women at high risk of preeclampsia and obstetric antiphospholipid syndrome to prevent diverse pathologies of gestation 53 . Nonetheless, aspirin administration for breast cancer prevention or its treatment is still debatable. So far, plenty of clinical trials have explored the effects of aspirin in breast cancer patients 54 . A recent updated meta-analysis of 38 observational studies yielded that aspirin could reduce the risk of breast cancer patients, such as postmenopausal, hormone receptor-positive tumors, or in situ tumors 55 . Although pooled observational studies have shown that long-term aspirin usage is associated with a low risk of breast cancer incidence, a recent meta-analysis of clinical trials showed that aspirin did not necessarily reduce cancer risk (RR = 1.01, 95% CI: 0.97-1.04). The discrepancies between the clinical and observational studies may reduce the potential clinical practicality of aspirin in breast cancer management. Notwithstanding this dispute, recent clinical trials where aspirin was used as adjuvant therapy or as an add-on strategy showed promising results. According to Joharatnam-Hogan et al. (2019), the regular use of aspirin after standard treatments could prevent recurrence and prolong survival in breast cancer patients 56 . Our results showed that the aspirin users had better expressional biomarkers than the non-aspirin users. Although both groups showed significant improvements in their expressional profiles, these changes were more prominent in aspirin users. These findings suggest that aspirin can increase the efficacy of current chemotherapies by increasing the sensitivity of the cancer cells to chemotherapy. Interestingly, we could demonstrate this benefit of aspirin in the clinical outcomes such as 5-year overall survival. We followed our patients for a long time and showed that the patients exhibited a longer overall survival rate after aspirin consumption than the non-aspirin group. Similar to our findings, Liue et al. (2021) showed that aspirin reduced breast-cancer-specific death by 31%, and the risk of recurrence/metastasis decreased by 9%. In this respect, aspirin may improve all-cause mortality, specific mortality, and risk of recurrence/metastasis in patients with breast cancer 57 . Sendur et al. (2014) also showed that despite the contradictory results regarding aspirin and breast cancer incidence, its use in breast cancer was associated with improved disease-free survival. Aspirin users had a significantly lower incidence of histological grade II-III tumors, but no effect was found on other clinicopathological properties 58 .

Conclusion
We demonstrated that adding aspirin to the treatment of breast cancer patients could reduce the expression of oncolncRNAs, oncomiRs, and oncogenes and simultaneously increase the levels of tumor-suppressor lncRNAs, miRs, and mRNAs.
The analysis of GO term pathways by the FunRich software. The pathway enrichment analyses of the GO database were executed through the FunRich (http:// www. funri ch. org) software 18 . Likewise, the same genes were explored for pathway enrichment using the g: Profiler tool (http:// biit. cs. ut. ee/ gprofi ler) 59 . At last, the miR/target gene regulatory network was built using the Cytoscape (https:// cytos cape. org) software 59 .
LncRNA-miR-mRNA network construction. The lncRNA-miR-mRNA network was constructed and visualized using Cytoscape software based on the ceRNA theory 59 . Here, the nodes and edges represent extensive biological data described previously 18 . A network analysis was performed using a Cytoscape plug-in to explore the structure and feature of the lncRNA-miR-mRNA competing triplets 59 . Sample collection. This study is part of an ongoing randomized clinical trial registered in the Iranian randomized control trial (IRCT2016080818745N11). All participants were informed of the current research objectives, study protocol, and informed consent to participate in the study. The proposal was approved by the Ethics   18 . Besides, ten normal-risk women who had attended the breast clinic for screening purposes and had healthy breasts were entered as the controls. The aim and protocol of the study were explained to all participants, and they all provided written informed consent. The right to withdraw from the survey was reserved for all patients at any time. www.nature.com/scientificreports/ In all patients with breast cancer, 10 cc of blood was withdrawn twice at a three-month interval at the point of entry and at the end of the study period for each participant (defined below). The blood was centrifuged at 3000 g for 5 min, and the plasma was preserved at − 80 °C. The patients' characteristics included age, tumor size, nodal status, histologic type, Her2, Ki-67, and hormone receptor status 62 . Inclusion criteria for patients with breast cancer. Exclusion criteria for patients with breast cancer.
Regional lymph node involvement Evidence of distant metastasis Pregnancy or breastfeeding Prior long-term aspirin use History of sensitivity to aspirin Platelet count < 100,000/µL History of coagulopathy or use of anti-coagulative agents  www.nature.com/scientificreports/ bers. The allocation of treatments was performed in a 1:1 ratio, and the treatments were assigned using a sealed envelope. The oncologist in charge of the chemotherapy and the patients themselves knew about the groupings (except for the surgeons responsible for the patient and the researchers who collected the blood samples and performed the molecular and cellular tests) were blind to it. According to histologic results of the surgical specimens after the operation and the oncologist's decision, several patients did not need chemotherapy and only underwent endocrine therapy as their systemic adjuvant treatment; these patients were withdrawn from the study. The other patients received their chemotherapy regimen of adriamycin, cyclophosphamide, and a taxane.
Interventions. The patients in the Aspirin group received an oral daily dose of 80 mg over three months (Fig. 11). Aspirin administration was initiated after the operation during chemotherapy and continued for three months throughout chemotherapy in all patients. The non-aspirin group received no aspirin or other NSAID during the first three months of the chemotherapy.  www.nature.com/scientificreports/ Primary outcomes. We measured the expression of the lncRNAs (Table 5), miRs (Table 1), and mRNAs (Table 4) as the primary outcomes before (baseline) and after three months of the intervention in the Aspirin and non-aspirin groups.
Secondary outcomes. We evaluated the protein levels of TGFβ, IFNγ, IL-17, and IL-1β pre-and-post intervention (Table 7) as the secondary outcomes before (baseline) and after three months of the intervention in the Aspirin and non-aspirin groups.
Real-time PCR analysis. The RNA was extracted from the plasma samples. Plasma (250 μl) was added to 750 μl TRIzol (Beijing Tiangen Biotech Co., Ltd.). RNA extraction was then carried out according to the manu-  TGFBR2  GCT TTG CTG AGG TCT ATA AGGC  GGT ACT CCT GTA GGT TGC CCT   PIK3CD  TGG CGG ATA GAC ATA CAT TGC  ACC AGT AGG CAA CCG TGA AG   AKT3  TGA AGT GGC ACA CAC TCT AACT  CCG CTC TCT CGA CAA ATG GA   ERBB2  CAG GGG TGG TAT TGT TCA GC  GGG AAA CCT GGA ACT CAC CT   SOCS5  TGA GCC TAC CAC ACG GTA TTATG  GAT TGT ACT TAC TCA ATG ACCT   IGF1  GCT CTT CAG TTC GTG TGT GGA  GCC TCC TTA GAT CAC AGC TCC   MYC  GAC CAG AAA AGT AGC TGC CG  GCC CGG ATG TGC ACT AAA AT   NOTCH1  ACA GTC TGG GCC TAT GAA ACC  TGT GAA CGT GAT GTC AAC GAG   PTEN  GGT GGG TTA TGG TCT TCA AAAGG  TGG ATT CGA CTT AGA CTT GACCT   FOXO3  CAC GGC TTG CTT ACT GAA GG  TCA CGC ACC AAT TCT AAC GC   B-actin  CAC CAT TGG CAA TGA GCG GTTC  AGG TCT TTG CGG ATG TCC  www.nature.com/scientificreports/ facturer's instructions. The absorbance ratio (A260/280) of total RNA, between 1.8 and 2.2, was determined using an ultraviolet (UV) spectrophotometer. According to the manufacturer's recommendations, the miRcute miRNA cDNA First-Strand Synthesis kit (Beijing Tiangen Biotech Co., Ltd.) for miRs quantification and the cDNA Synthesis Kit Manual (TAKARA BIO INC. Cat. 6 30 v.0708) for mRNAs and lncRNAs quantification were used. Then, cDNA was used in each real-time PCR assay with the miRcute miR Fluorescence Quantitative Detection kit (Tiangen Biotech Co., Ltd.) for miRs. The cycling conditions were the pre-denaturation at 94 °C for 2 min, followed by 40 cycles of 94 °C for 20 s and 60 °C for 34 s. The SYBR Green method (AccuPower Green Star qPCR Master Mix; Bioneer, Korea) was used for genes and lncRNAs. PCR cycling was performed as follows: one cycle at 95 °C for 10 min, 40 cycles at 95 °C for 20 s, and 60 °C for 45 s. The melting curve analysis was run from 60 to 95 °C to confirm specific amplification 18,59 . The expression of U6 and B-actin was used to normalize miRs, lncRNAs, and genes as the Internal Reference Gene. The list of primers has shown in Table 8. The qRT-PCR reactions were performed using an ABI StepOne plus System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The expression level of the genes was calculated using the − ΔCT method. ΔCT was calculated by subtracting the CT values of U6 and B-actin from the targets 63,64 .
Clinical outcomes. We evaluated the association between the intervention groups with the clinicopathological feature of patients, such as the 5-year overall survival.
Data analysis. The sample size was calculated based on a study by Chen et al. (2016) and the differences in miR-21 expression in healthy subjects and patients with breast cancer 65 . The sample size was 23 in each arm, considering the alpha error less than 0.05 (α) and the research power of 95% (1-β). The data analyses were performed by GraphPad Prism 7.0 (https:// www. graph pad. com). We used the t-test and the Mann-Whitney to analyze the parametric and non-parametric data in two groups. The 5-year overall survival rate was evaluated using the Kaplan-Meier method. All data were presented as mean ± SD. P-value < 0.05 was considered to be statistically significant.
Ethical approval. The experimental procedures and care protocols were approved by a review board committee of Tehran University of Medical Sciences (No: IR.TUMS.VCR.REC.1397.606) and registered by the Iranian Randomized Control Trial (IRCT) ethical board (No: IRCT2016080818745N11). Written informed consent was obtained from each participant before the sample collection.